This invention relates to plant genetic engineering. In particular, it relates to methods of modulating transcription in plant cells.
An array of eukaryotic functions are regulated at the transcriptional level by a type of DNA-binding proteins encoded by the MYB-domain genes (Martin, C. et al., Trends in Genet 13:67-73 (1997); Thompson, M. A. et al., Bioessays 17:341-350 (1995)). MYB proteins are characterized by a modular design, comprising discrete functional domains that permit transcription activities to be highly regulated. The amino-terminal DNA-binding domain, or DBD, consists of two or three helix-tum-helix motifs of 51-52 amino acids (R1, R2 and R3) that are highly conserved across phyla. Yet, the precise structure of each of the DBDs determines the specificity of MYB-DNA interactions, and in turn, dictates the level of MYB-mediated transcription (Ramsay, R. G. et al., J. Biol Chem 267:5656-5662 (1992); Tanikawa, J. et al., Proc Natl Acad Sci USA 90:9320-9324 (1993)). The transactivation domain, or TAD, varies in composition and in its relative position within the protein from MYB-to-MYB (Paz-Ares, J. et al., EMBO J 9:315-321 (1990); Sainz, M. B. et al., Mol Cell Biol 17:115-122 (1997); Urao, T. et al., Plant J 10:1145-1148 (1996)), and serves to regulate transcription efficiency in trans. A leucine-zipperlike structure that presumably mediates MYB-MYB interactions, as well as protein interactions with other transcription factors (Kanei-Ishii, C. et al., Proc Natl Acad Sci USA 89:3088-3092 (1992); Nomura, T. et al., J Biol Chem 268:21914-21923 (1993)) is referred to as the negative regulatory domain (NRD). However, NRDs have thus far only been identified in animal systems. MYB-mediated transcription is also subject to modulation by the transcription and translation rates inherent to the MYB genes themselves (Nicolaides, N. C. et al., J Biol Chem 267:19665-19672 (1992); Wissenbach, M. et al., Plant J 4:411-422 (1993)).
In contrast to other eukaryotes which contain only a few copies per haploid genome (Thompson, M. A. et al., Bioessays 17:341-350 (1995)), the number of genes in the R2R3-MYB family in plant genomes is considerably higher (Avila, J. et al., Plant J 3:553-562 (1993); Jackson, D. et al., Plant Cell 3:115-125 (1991); Lin, Q. et al., Plant Mol Biol 30:1009-1020 (1996); Lipsick, J. S. Oncogene 13:223-235 (1996); Romero, L. et al., Plant J 14:273-284 (1998); Solano, R. et al., Plant J 8:673-682 (1995b)). At least 85 R2R3-MYB genes have been identified in Arabidopsis thaliana thus far (Romero, L. et al., Plant J 14:273-284 (1998); Meissner et al., Plant Cell. 10:1827-40 (1999)). The expansion of the plant R2R3-MYB gene family during the course of evolution is believed by many to provide a mechanism for the regulation of plant-specific processes and functions (Martin, C. et al., Trends in Genet 13:67-73 (1997)). Most of the relatively few plant MYBs that have been assigned functions are involved in regulation of phenylpropanoid biosynthesis (Cone, K. C. et al., Plant Cell 5:1795-1805 (1993); Franken, P. et al., Plant J 6:21-30 (1994); Grotewold, E. et al., Cell 76:543-553 (1994); Moyano, E. et al., Plant Cell 8:1519-1532 (1996); Quattrocchio et al., Plant J 13:475-488 (1993); Solano et al., EMBO J. 14:1773-1784 (1995)). In two known instances, MYB genes control the differentiation of epidermal cells. Glabrous1 (AtMYBG/1) governs leaf trichome formation in Arabidopsis thaliana (Oppenheimer, D.G. et al., Cell 67:483-493 (1991)), while MIXTA (AmMYBMx) of Antirrhinum majus controls the development of conical cells or multicellular trichomes, depending on the timing of MIXTA gene expression (Glover, B. J. et al., Development 125:3497-3508 (1998)).
The economically important xe2x80x9cfibersxe2x80x9d of cotton used in textile manufacturing are, in actuality, single-celled seed trichomes that develop from the epidernis of the ovule (Wilkins, T. A. et al., In Basra AS (ed) Cotton Fibers. Food Products Press New York (1999)). There is a need to improve the quality of cotton fibers for use in a variety of textile products, In particular, means for improving fiber, such as fiber strength, fiber length and the like. The present invention addresses these and other needs.
The present invention provides methods of modulating transcription in a plants. The methods comprise introducing into a plant a recombinant expression cassette comprising a promoter sequence operably linked to a heterologous polynucleotide sequence encoding a MYB polypeptide. A MYB polypeptide of the invention can be, for example, a polypeptide that is at least substantially identical to MYB poylypeptides exemplified here (e.g. SEQ ID NOS:2, 4, 6 or 8). The polynucleotide can be, for example, SEQ ID NOS:1, 3, 5, or 7.
The particular plant used in the methods of the invention is not critical. In some embodiments, the plant is a cotton plant. In these embodiments, it is particularly useful to use a promoter that directs expression of the polynucleotide sequence in cotton fibers.
A explained below, a number of valuable phenotypes are conferred on plants produced by the methods of the invention. They include, for examnple, increased fiber quality, alteration of root architecture, enhanced growth and the like. A recombinant expression cassette comprising a promoter sequence operably linked to a heterologous polynucleotide sequence encoding a MYB polypeptide.
The invention further provides recombinant expression cassettes useful in the methods of the invention. Plants made by the claimed methods are also provided.
The phrase xe2x80x9cnucleic acid sequencexe2x80x9d refers to a single or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5xe2x80x2 to the 3xe2x80x2 end. It includes chromosomal DNA, self-replicating plasmids, infectious polymers of DNA or RNA and DNA or RNA that performs a primarily structural role.
A xe2x80x9cpromoterxe2x80x9d is defined as an array of nucleic acid control sequences that direct transcription of an operably linked nucleic acid. As used herein, a xe2x80x9cplant promoterxe2x80x9d is a promoter that functions in plants, even though obtained from other organisms, such as plant viruses. Promoters include necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter also optionally includes distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription. The term xe2x80x9coperably linkedxe2x80x9d refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter, or array of transcription factor binding sites) and a second nucleic acid sequence, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.
The term xe2x80x9cplantxe2x80x9d includes whole plants, plant organs (e.g., leaves, stems, flowers, roots, etc.), seeds and plant cells and progeny of same. The class of plants that can be used in the method of the invention is generally as broad as the class of higher plants amenable to transformation techniques, including angiosperms (monocotyledonous and dicotyledonous plants), as well as gymnosperms. It includes plants of a variety of ploidy levels, including polyploid, diploid, haploid and hemizygous.
A polynucleotide sequence is xe2x80x9cheterologous toxe2x80x9d an organism or a second polynucleotide sequence if it originates from a foreign species, or, if from the same species, is modified from its original form. For example, a promoter operably linked to a heterologous coding sequence refers to a coding sequence from a species different from that from which the promoter was derived, or, if from the same species, a coding sequence which is different from any naturally occurring allelic variants.
A polynucleotide xe2x80x9cexogenous toxe2x80x9d an individual plant is a polynucleotide which is introduced into the plant by any means other than by a sexual cross. Examples of means by which this can be accomplished are described below, and include Agrobacterium-mediated transformation, particle-mediated methods, electroporation, and the like. Such a plant containing the exogenous nucleic acid is referred to here as an R1 generation transgenic plant. Transgenic plants that arise from sexual cross or by selfing are descendants of such a plant.
The term xe2x80x9cMYB polynucleotidexe2x80x9d refers to a polynucleotides encoding a member of a class of transcription factors referred to here as xe2x80x9cMYB polypeptidesxe2x80x9d. MYB polypeptides are characterized by the presence of an amino-terminal DNA-binding domain, or DBD, consisting of two or three helix-turn-helix motifs of 51-52 amino acids (R1, R2 and R3) that are highly conserved across phyla. MYB polypeptides may also comprise a transactivation domain. Exemplary MYB polypeptides are disclosed in SEQ ID NO:1 (GhMYB 1 GenBank Accession No. L04497) and SEQ ID NO:3 (GhMYB 6 GenBank Accession No. AF034134). Other useful sequences include sequences at GenBank Nos. AF034130 (GhMYB 2), AF034131 (GhMYB 3), AF034132 (GhMYB 4), and AF034133 (GhMYB 5). In addition, two other MYB nucleotide sequences are provided (GhMYB 7 and 8 (SEQ ID NOS:5 and 7). One of skill in the art will recognize that in light of the present disclosure, various modifications (e.g., substitutions, additions, and deletions) can be made to the MYB polypeptide sequences without substantially affecting their function. For example, the MYB polypeptides may contain functional domains from other porteins (e.g. related MYB polypeptides). These variations are within the scope of the term xe2x80x9cMYB polypeptidexe2x80x9d. For example a MYB polypeptide includes the sequences exemplified here as well as polypeptides that are at least about 60%, usually at least about 70%, more usually at least about 80%, and often at least about 90% identical to the exemplified sequences. Also included are variant nucleic acid sequences that encode the same polypeptide as the exemplified sequences, i.e. sequences comprising degenerate sequences.
In the case of both expression of transgenes and inhibition of endogenous genes (e.g., by antisense, or sense suppression) one of skill will recognize that the inserted polynucleotide sequence need not be xe2x80x9cidentical,xe2x80x9d but may be only xe2x80x9csubstantially identicalxe2x80x9d to a sequence of the gene from which it was derived.
The terms xe2x80x9cidenticalxe2x80x9d or percent xe2x80x9cidentity,xe2x80x9d in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection.
The phrase xe2x80x9csubstantially identical,xe2x80x9d in the context of two nucleic acids or polypeptides, refers to two or more sequences or subsequences that have at least about 60%, or at least about 70%, preferably at least about 80%, most preferably at least about 90-95% nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection. Preferably, the substantial identity exists over a region of the sequences that is at least about 50 residues in length, more preferably over a region of at least about 100 residues, and most preferably the sequences are substantially identical over at least about 150 residues. In a most preferred embodiment, the sequences are substantially identical over the entire length of the coding regions.
For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.
Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Nat""l. Acad. Sci. USA 85:2444(1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally, Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley and Sons, Inc., (1995 Supplement) (Ausubel)).
Examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and Altschuel et al. (1977) Nucleic Acids Res. 25: 3389-3402, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative aligrnent score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always greater than 0) and N penalty score for mismatching residues; always less than 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=xe2x88x924, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).
In addition to calculating percent sequence identity, the BLAST algorithmn also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Nat""l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.
A further indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the polypeptide encoded by the second nucleic acid. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions, as described below.
xe2x80x9cConservatively modified variantsxe2x80x9d applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are xe2x80x9csilent variations,xe2x80x9d which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence.
As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a xe2x80x9cconservatively modified variantxe2x80x9d where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing finctionally similar amino acids are well known in the art.
The following six groups each contain amino acids that are conservative substitutions for one another:
1) Alanine (A), Serine (S), Threonine (T);
2) Aspartic acid (D), Glutamic acid (E);
3) Asparagine (N), Glutamine (Q);
4) Arginine (R), Lysine (K);
5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and
6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).
(see, e.g., Creighton, Proteins (1984)).
An indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the antibodies raised against the polypeptide encoded by the second nucleic acid. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below.
The phrase xe2x80x9cselectively (or specifically) hybridizes toxe2x80x9d refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent hybridization conditions when that sequence is present in a complex mixture (e.g., total cellular or library DNA or RNA).
The phrase xe2x80x9cstringent hybridization conditionsxe2x80x9d refers to conditions under which a probe will hybridize to its target subsequence, typically in a complex mixture of nucleic acid, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biologyxe2x80x94Hybridization with Nucleic Probes, xe2x80x9cOverview of principles of hybridization and the strategy of nucleic acid assaysxe2x80x9d (1993). Generally, highly stringent conditions are selected to be about 5-10xc2x0 C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength pH. Low stringency conditions are generally selected to be about 15-30xc2x0 C. below the Tm. The Tm is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at Tm, 50% of the probes are occupied at equilibrium). Stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30xc2x0 C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60xc2x0 C. for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as forrnnide. For selective or specific hybridization, a positive signal is at least two times background, preferably 10 time background hybridization.
Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides which they encode are substantially identical. This occurs, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. In such cased, the nucleic acids typically hybridize under moderately stringent hybridization conditions.
In the present invention, genomic DNA or cDNA comprising nucleic acids of the invention can be identified in standard Southern blots under stringent conditions using the nucleic acid sequences disclosed here. For the purposes of this disclosure, suitable stringent conditions for such hybridizations are those which include a hybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37xc2x0 C., and at least one wash in 0.2xc3x97SSC at a temperature of at least about 50xc2x0 C., usually about 55xc2x0 C. to about 60xc2x0 C., for 20 minutes, or equivalent conditions. A positive hybridization is at least twice background. Those of ordinary skill will readily recognize that alternative hybridization and wash conditions can be utilized to provide conditions of similar stringency.
A further indication that two polynucleotides are substantially identical is if the reference sequence, amplified by a pair of oligonucleotide primers, can then be used as a probe under stringent hybridization conditions to isolate the test sequence from a cDNA or genomic library, or to identify the test sequence in, e.g., a northern or Southern blot.
xe2x80x9cFiber specificxe2x80x9d promoter refers to promoters that preferentially promote gene expression in fiber cells over other cell types.
This invention provides methods of using MYB transcription factors to modulate transcription plant cells and there by modify plant phenotypes. Of particular interest to the present invention is the use of these polynucleotides to modulate cotton fiber yield and quality. The transcription factors of the invention are also useful in modulating plant architecture and morphology as well as development and time to flowering. The polynucleotides of the invention can also be targeted to root cells and used to modulate root architecture and biomass. In particular, the polynucleotides can be used to increase the number and length of root hairs.
The present invention is based, at least in part, on experiments designed to determine the degree to which MYBs are involved in controlling the differentiation, growth and development of cotton seed trichomes. A cotton ovule cDNA library was screened using a PCR-based strategy and AtMYBG/1 as a heterologous hybridization probe. Six MYB genes, designated as GhMYB1 through GhMYB6, were identified from cotton ovules. However, apart from the expected conservation of the DBD, none of the cotton MYBs showed any striking similarity to Glabrous1 or MIXTA. Analysis of the spatial and temporal regulation of GhMYBs in different tissue-types and during fiber development revealed two general patterns of gene expression. One group of GhMYB genes (type I) are relatively more abundant and appear to be expressed in all tissues examined, whereas transcripts of the second group (type H) are less-abundant than type I and exhibit tissue-specific patterns of expression. Despite the lack of overall similarity to Glabrous1 and MIXTA, developmentally-regulated expression of the cotton R2R3-MYB genes is stage-specific and consistent with a functional role in cotton trichome differentiation and expansion (see, Loguercio et al. Mol. Gen. Genet 261:660-671 (1999)).
Isolation of Nucleic Acids
Generally, the nomenclature and the laboratory procedures in recombinant DNA technology described below are those well known and commonly employed in the art. Standard techniques are used for cloning, DNA and RNA isolation, amplification and purification. Generally enzymatic reactions involving DNA ligase, DNA polymerase, restriction endonucleases and the like are performed according to the manufacturer""s specifications. These techniques and various other techniques are generally performed according to Sambrook et al., Molecular Cloningxe2x80x94A Laboratory Manual, Cold Spring Habor Laboratory Cold Spring Harbor, N.Y., (1989) or Current Protocols in Molecular Biology Volumes 1-3, John Wiley and Sons, Inc. (1994-1998).
The isolation of nucleic acids may be accomplished by a number of techniques. For instance, oligonucleotide probes based on the sequences disclosed here can be used to identify the desired gene in a cDNA or genomic DNA library. To construct genomic libraries, large segments of genomic DNA are generated by random fragmentation, e.g. using restriction endonucleases, and are ligated with vector DNA to form concatemers that can be packaged into the appropriate vector. To prepare a cDNA library, mRNA is isolated from the desired organ, such as leaves, and a cDNA library which contains gene transcripts is prepared from the mRNA. Alternatively, cDNA may be prepared from mRNA extracted from other tissues in which genes of interest or their homologs are expressed.
The cDNA or genomic library can then be screened using a probe based upon the sequence of a cloned gene disclosed here. Probes may be used to hybridize with genomic DNA or cDNA sequences to isolate homologous genes in the same or different plant species. Alternatively, antibodies raised against a polypeptide of interest can be used to screen an mRNA expression library.
Alternatively, the nucleic acids of interest can be amplified from nucleic acid samples using amplification techniques. For instance, polymerase chain reaction (PCR) technology can be used to amplify the sequences of genes directly from genomic DNA, from cDNA, from genomic libraries or cDNA libraries. PCR and other in vitro amplification methods may also be useful, for example, to clone nucleic acid sequences that code for proteins to be expressed, to make nucleic acids to use as probes for detecting the presence of the desired mRNA in samples, for nucleic acid sequencing, or for other purposes. For a general overview of PCR, see PCR Protocols: A Guide to Methods and Applications. (Innis, M, Gelfand, D., Sninsky, J. and White, T., eds.), Academic Press, San Diego (1990). Appropriate primers and probes for identifing sequences from plant tissues are generated from comparisons of the sequences provided herein (e.g. SEQ ID NOS:1 and 3).
Polynucleotides may also be synthesized by well-known techniques, as described in the technical literature. See, e.g., Carruthers et al., Cold Spring Harbor Symp. Quant. Biol. 47:411-418 (1982), and Adams et al., J. Am. Chem. Soc. 105:661 (1983). Double stranded DNA fragments may then be obtained either by synthesizing the complementary strand and annealing the strands together under appropriate conditions, or by adding the complementary strand using DNA polymerase with an appropriate primer sequence.
Increase Levels of Gene Expression in Plant Fibers
The isolated nucleic acid sequences prepared as described herein can be used in a number of techniques. For example, the isolated nucleic acids can be introduced into plants to enhance endogenous MYB gene expression and thereby increase expression of the genes whose expression is controlled by MYB polypeptides. A particularly useful gene for this purpose are the MYB genes shown in SEQ ID NO: 1, and 3.
The isolated nucleic acid sequences prepared as described herein can be used in a number of techniques. For example, the isolated nucleic acids can be introduced into plants to enhance endogenous MYB gene expression and thereby increase expression of the genes whose expression is controlled by MYB polypeptides. A particularly useful gene for this purpose are the MYB genes shown in SEQ ID NOS:1 and 3.
Modified protein chains can also be readily designed utilizing various recombinant DNA techniques well known to those skilled in the art and described in detail below. For example, the chains can vary from the naturally occurring sequence at the primary structure level by amino acid substitutions, additions, deletions, and the like. These modifications can be used in a number of combinations to produce the final modified protein chain.
In another embodiment, endogenous gene expression can be targeted for modification. Methods for introducing genetic mutations into plant genes and selecting plants with desired traits are well known. For instance, seeds or other plant material can be treated with a mutagenic chemical substance, according to standard techniques. Such chemical substances include, but are not limited to, the following: diethyl sulfate, ethylene imine, ethyl methanesulfonate and N-nitroso-N-ethylurea. Alternatively, ionizing radiation from sources such as X-rays or gamma rays can be used.
Alternatively, homologous recombination can be used to induce targeted gene modifications by specifically targeting the MYB gene in vivo (see, generally, Grewal and Klar, Genetics 146: 1221-1238 (1997) and Xu et al., Genes Dev. 10: 2411-2422 (1996)). Homologous recombination has been demonstrated in plants (Puchta et al., Experientia 50: 277-284 (1994), Swoboda et al., EMBO J. 13: 484-489 (1994); Offringa et al., Proc. Natl. Acad. Sci. USA 90: 7346-7350 (1993); and Kempin et al., Nature 389:802-803 (1997)).
In applying homologous recombination technology to the genes of the invention, mutations in selected portions of a MYB gene sequence (including 5xe2x80x2 upstream, 3xe2x80x2 downstream, and intragenic regions) such as those disclosed herein are made in vitro and then introduced into the desired plant using standard techniques. Since the efficiency of homologous recombination is known to be dependent on the vectors used, use of dicistronic gene targeting vectors as described by Mountford et al., Proc. Natl. Acad. Sci. USA 91: 4303-4307 (1994); and Vaulont et al., Transgenic Res. 4: 247-255: (1995) are conveniently used to increase the efficiency of selecting for altered MYB expression in transgenic plants. The mutated gene will interact with the target wild-type gene in such a way that homologous recombination and targeted replacement of the wild-type gene will occur in transgenic plant cells, resulting in increased MYB activity.
Alternatively, oligonucleotides composed of a contiguous stretch of RNA and DNA residues in a duplex conformation with double hairpin caps on the ends can be used. The RNA/DNA sequence is designed to align with the sequence of the target gene and to contain the desired nucleotide change. Introduction of the chimeric oligonucleotide on an extrachromosomal T-DNA plasmid results in efficient and specific MYB gene conversion directed by chimeric molecules in a small number of transformed plant cells. This method is described in Cole-Strauss et al., Science 273:1386-1389 (1996) and Yoon et al., Proc. Natl. Acad Sci. USA 93: 2071-2076 (1996).
One method to increase activity of desired gene products is to use xe2x80x9cactivation mutagenesisxe2x80x9d (see, e.g., Hiyashi et al. Science 258:1350-1353 (1992)). In this method an endogenous gene can be modified to be expressed constitutively, ectopically, or excessively by insertion of T-DNA sequences that contain strong/constitutive promoters upstream of the endogenous gene. Activation mutagenesis of the endogenous gene will give the same effect as overexpression of the transgenic nucleic acid in transgenic plants. Alternatively, an endogenous gene encoding an enhancer of gene product activity or expression of the gene can be modified to be expressed by insertion of T-DNA sequences in a similar manner and MYB activity can be increased.
Another strategy to increase gene expression can involve the use of dominant hyperactive mutants of the gene by expressing modified transgenes. For example, expression of a modified MYB with a defective domain that is important for interaction with a negative regulator of MYB activity can be used to generate dominant hyperactive MYB proteins. Alternatively, expression of truncated MYB which have only a domain that interacts with a negative regulator can titrate the negative regulator and thereby increase endogenous MYB activity. Use of dominant mutants to hyperactivate target genes is described, e.g., in Mizukami et al., Plant Cell 8:831-845 (1996).
Supression of MYB Expression
The nucleic acid sequences disclosed here can be used to design nucleic acids useful in a number of methods to inhibit MYB or related gene expression in plants. For instance, antisense technology can be conveniently used. To accomplish this, a nucleic acid segment from the desired gene is cloned and operably linked to a promoter such that the antisense strand of RNA will be transcribed. The construct is then transformed into plants and the antisense strand of RNA is produced. In plant cells, it has been suggested that antisense suppression can act at all levels of gene regulation including suppression of RNA translation (see, Bourque Plant Sci. (Limerick) 105: 125-149 (1995); Pantopoulos In Progress in Nucleic Acid Research and Molecular Biology, Vol. 48. Cohn, W. E. and K. Moldave (Ed.). Academic Press, Inc.: San Diego, Calif. USA; London, England, UK. p. 181-238; Heiser et al. Plant Sci. (Shannon) 127: 61-69 (1997)) and by preventing the accumulation of mRNA which encodes the protein of interest, (see, Baulcombe Plant Mol. Bio. 32:79-88 (1996); Prins and Goldbach Arch. Virol. 141: 2259-2276 (1996); Metzlaff et al. Cell 88: 845-854 (1997), Sheehy et al., Proc. Nat. Acad. Sci. USA, 85:8805-8809 (1988), and Hiatt et al., U.S. Pat. No. 4,801,340).
The nucleic acid segment to be introduced generally will be substantially identical to at least a portion of the endogenous MYB gene or genes to be repressed. The sequence, however, need not be perfectly identical to inhibit expression. The vectors of the present invention can be designed such that the inhibitory effect applies to other genes within a family of genes exhibiting identity or substantial identity to the target gene.
For antisense suppression, the introduced sequence also need not be full length relative to either the primary transcription product or fully processed mRNA. Generally, higher identity can be used to compensate for the use of a shorter sequence. Furthermore, the introduced sequence need not have the same intron or exon pattern, and identity of non-coding segments may be equally effective. Normally, a sequence of between about 30 or 40 nucleotides and about full length nucleotides should be used, though a sequence of at least about 100 nucleotides is preferred, a sequence of at least about 200 nucleotides is more preferred, and a sequence of about 500 to about 3500 nucleotides is especially preferred.
A number of gene regions can be targeted to suppress MYB gene expression. The targets can include, for instance, the coding regions, introns, sequences from exon/intron junctions, 5xe2x80x2 or 3xe2x80x2 untranslated regions, and the like.
Another well known method of suppression is sense co-suppression. Introduction of nucleic acid configured in the sense orientation has been recently shown to be an effective means by which to block the transcription of target genes. For an example of the use of this method to modulate expression of endogenous genes (see, Assaad et al., Plant Mol. Bio. 22: 1067-1085 (1993); Flavell Proc. Natl. Acad. Sci. USA 91: 3490-3496 (1994); Stam et al. Annals Bot. 79: 3-12 (1997); Napoli et al., The Plant Cell 2:279-289 (1990); and U.S. Pat. Nos. 5,034,323, 5,231,020, and 5,283,184).
The suppressive effect may occur where the introduced sequence contains no coding sequence per se, but only intron or untranslated sequences homologous to sequences present in the primary transcript of the endogenous sequence. The introduced sequence generally will be substantially identical to the endogenous sequence intended to be repressed. This minimal identity will typically be greater than about 65%, but a higher identity might exert a more effective repression of expression of the endogenous sequences. Substantially greater identity of more than about 80% is preferred, though about 95% to absolute identity would be most preferred. As with antisense regulation, the effect should apply to any other proteins within a similar family of genes exhibiting identity or substantial identity.
For co-suppression, the introduced sequence, needing less than absolute identity, also need not be full length, relative to either the primary transcription product or fully processed mRNA. This may be preferred to avoid concurrent production of some plants which are overexpressers. A higher identity in a shorter than full length sequence compensates for a longer, less identical sequence. Furthermore, the introduced sequence need not have the same intron or exon pattern, and identity of non-coding for antisense regulation is used. In addition, the same gene regions noted for antisense regulation can be targeted using co-suppression technologies.
Oligonucleotide-based triple-helix formation can also be used to disrupt MYB gene expression. Triplex DNA can inhibit DNA transcription and replication, generate site-specific mutations, cleave DNA, and induce homologous recombination (see, e.g., Havre and Glazer J. Virology 67:7324-7331 (1993); Scanlon et al. FASEB J 9:1288-1296 (1995); Giovannangeli et al. Biochemistry 35:10539-10548 (1996); Chan and Glazer J. Mol. Medicine (Berlin) 75: 267-282 (1997)). Triple helix DNAs can be used to target the same sequences identified for antisense regulation.
Catalytic RNA molecules or ribozyrnes can also be used to inhibit expression of MYB genes. It is possible to design ribozymes that specifically pair with virtually any target RNA and cleave the phosphodiester backbone at a specific location, thereby functionally inactivating the target RNA. In carrying out this cleavage, the ribozyme is not itself altered, and is thus capable of recycling and cleaving other molecules, making it a true enzyrne. The inclusion of ribozyme sequences within antisense RNAs confers RNA-cleaving activity upon them, thereby increasing the activity of the constructs. Thus, ribozymes can be used to target the same sequences identified for antisense regulation.
A number of classes of ribozymes have been identified. One class of ribozymes is derived from a number of small circular RNAs which are capable of self-cleavage and replication in plants. The RNAs replicate either alone (viroid RNAs) or with a helper virus (satellite RNAs). Examples include RNAs from avocado sunblotch viroid and the satellite RNAs from tobacco ringspot virus, lucerne transient streak virus, velvet tobacco mottle virus, solanum nodiflorum mottle virus and subterranean clover mottle virus. The design and use of target RNA-specific ribozymes is described in Zhao and Pick, Nature 365:448-451 (1993); Eastham and Ahlering, J. Urology 156:1186-1188 (1996); Sokol and Murray, Transgenic Res. 5:363-371 (1996); Sun et al., Mol. Biotechnology 7:241-251(1997); and Haseloff et al., Nature, 334:585-591 (1988).
Preparation of Recombinant Vectors
To use isolated sequences in the above techniques, recombinant DNA vectors suitable for transformation of plant cells are prepared. Techniques for transforming a wide variety of higher plant species are well known and described in the technical and scientific literaturee. for example, Weising et al., Ann. Rev. Genet. 22:421-477 (1988). A DNA sequence coding for the desired polypeptide, for example a cDNA sequence encoding a full length protein, will preferably be combined with transcriptional and translational initiation regulatory sequences which will direct the transcription of the sequence from the gene in the intended tissues of the transformed plant.
For example, for overexpression, a plant promoter fragment may be employed which will direct expression of the gene in all tissues of a regenerated plant. Such promoters are referred to herein as xe2x80x9cconstitutivexe2x80x9d promoters and are active under most environmental conditions and states of development or cell differentiation. Examples of constitutive promoters include the cauliflower mosaic virus (CAMa) 35S and 19S transcription initiation regions; the full-length FMV transcript promoter (Gowda et al., J Cell Biochem 13D:301; the 1xe2x80x2- or 2xe2x80x2- promoter derived from T-DNA of Agrobacterium tumefaciens, and other transcription initiation regions from various plant genes known to those of skill. Such promoters and others are described, e.g. in U.S. Pat. No. 5,880,330. Such genes include for example, ACT11 from Arabidopsis (Huang et al., Plant Mol. Biol. 33:125-139 (1996)), Cat3 from Arabidopsis (GenBank No. U43147, Zhong et al., Mol. Gen. Genet. 251:196-203 (1996)), the gene encoding stearoyl-acyl carrier protein desaturase from Brassica napus (Genbank No. X74782, Solocombe et al. Plant Physiol. 104:1167-1176 (1994)), GPc1 from maize (GenBank No. X15596, Martinez et al. J. Mol. Biol 208:551-565 (1989)), and Gpc2 from maize (GenBank No. U45855, Manjunath et al., Plant Mol. Biol. 33:97-112 (1997)).
Alternatively, the plant promoter may direct expression of a nucleic acid in a specific tissue, organ or cell type (i.e., tissue-specific promoters) or may be otherwise under more precise environmental or developmental control (i.e., inducible promoters). Examples of environmental conditions that may effect transcription by inducible promoters include anaerobic conditions, elevated temperature, the presence of light, or sprayed with chemicals/hormones. Numerous inducible promoters are known in the art, any of which can be used in the present invention. Such promoters include the yeast metallothionine promoter, which is activated by copper ions (see, e.g., Mett et al. (1993) PNAS 90:4567), the dexarnethasone-responsive promoter, In2-1 and In2-2, which are activated by substituted benzenesulfonamides, and GRE regulatory sequences, which are glucocorticoid-responsive (Schena et al., Proc. Natl. Acad. Sci. U.S.A. 88: 0421 (1991)).
Tissue-specific promoters can be inducible. Similarly, tissue-specific promoters may only promote transcription within a certain time frame of developmental stage within that tissue. Other tissue specific promoters may be active throughout the life cycle of a particular tissue. One of skill will recognize that a tissue-specific promoter may drive expression of operably linked sequences in tissues other than the target tissue. Thus, as used herein a tissue-specific promoter is one that drives expression preferentially in the target tissue or cell type, but may also lead to some expression in other tissues as well.
In preferred embodiments, promoters that drive fiber-specific expression of polynucleotides can be used. Such expression can be achieved under the control of the fiber-specific promoters described in U.S. Pat No. 5,495,070, incorporated herein by reference. Alternatively, promoters from genes expressed in primarily in roots, for example alcohol dehydrogenase, can be used.
If proper polypeptide expression is desired, a polyadenylation region at the 3xe2x80x2-end of the coding region should be included. The polyadenylation region can be derived from the natural gene, from a variety of other plant genes, or from T-DNA.
The vector comprising the sequences (e.g., promoters or coding regions) from genes of the invention will typically comprise a marker gene that confers a selectable phenotype on plant cells. For example, the marker may encode biocide resistance, particularly antibiotic resistance, such as resistance to kanarnycin, G418, bleomycin, hygromycin, or herbicide resistance; such as resistance to chlorosulfuron or Basta.
Production of Transgenic Plants
DNA constructs of the invention may be introduced into the genome of the desired plant host by a variety of conventional techniques. For example, the DNA construct may be introduced directly into the genomic DNA of the plant cell using techniques such as electroporation and microinjection of plant cell protoplasts, or the DNA constructs can be introduced directly to plant tissue using ballistic methods, such as DNA particle bombardment.
Microinjection techniques are known in the art and well described in the scientific and patent literature. The introduction of DNA constructs using polyethylene glycol precipitation is described in Paszkowski et al. Embo. J. 3:2717-2722 (1984). Electroporation techniques are described in Fromm et al. Proc. Natl. Acad. Sci. USA 82:5824 (1985). Ballistic transformation techniques are described in Klein et al. Nature 327: 70-73(1987).
Alternatively, the DNA constructs may be combined with suitable T-DNA flanking regions and introduced into a conventional Agrobacterium tumefaciens host vector. The virulence functions of the Agrobacterium tumefaciens host will direct the insertion of the construct and adjacent marker into the plant cell DNA when the cell is infected by the bacteria. Agrobacterium tumefaciens-mediated transformation techniques, including disarming and use of binary vectors, are well described in the scientific literature. See, for example Horsch et al., Science 233:496-498 (1984), and Fraley et al. Proc. Natl. Acad. Sci. USA 80:4803 (1983) and Gene Transfer to Plants, Potrykus, ed. (Springer-Verlag, Berlin 1995).
Transformed plant cells which are derived by any of the above transformation techniques can be cultured to regenerate a whole plant which possesses the transformed genotype and thus the desired phenotype such as increased fiber length, strength or fineness. Such regeneration techniques rely on manipulation of certain phytohormones in a tissue culture growth medium, typically relying on a biocide and/or herbicide marker that has been introduced together with the desired nucleotide sequences. Plant regeneration from cultured protoplasts is described in Evans et al., Protoplasts Isolation and Culture, Handbook of Plant Cell Culture, pp. 124-176, MacMillilan Publishing Company, N.Y., 1983; and Binding, Regeneration of Plants, Plant Protoplasts, pp. 21-73, CRC Press, Boca Raton, 1985. Regeneration can also be obtained from plant callus, explants, organs, or parts thereof. Such regeneration techniques are described generally in Klee et al. Ann. Rev. of Plant Phys. 38:467-486 (1987).
The nucleic acids of the invention can be used to confer desired traits on essentially any fiber producing plants. These plants include cotton plants (Gossypium arboreum, Gossypium herbaceum, Gossypium barbadense and Gossypium hirsutum), silk cotton tree (Kapok, Ceiba pentandra), desert willow, creosote bush, winterfal, balsa, ramie, kenaf, hemp (Cannabis sativa), roselle, jute, sisal abaca and flax.
One of skill will recognize that after the expression cassette is stably incorporated in transgenic plants and confirmed to be operable, it can be introduced into other plants by sexual crossing. Any of a number of standard breeding techniques can be used, depending upon the species to be crossed.
Using known procedures one of skill can screen for plants of the invention by detecting the increase or decrease of an mRNA or protein of interest in transgenic plants. Means for detecting and quantifying mRNAs or proteins are well known in the art.
Assessing Fiber Quality
Fibers produced from the transgenic plants transformed with MYB nucleic acids are compared to control fibers (e.g., fibers from native plants or plants transformed with marker nucleic acids) to determine the extent of modulation of fiber properties. Modulation of fiber properties, such as fiber length, strength, or fineness, is achieved when the percent difference in these fiber properties of transgenic plants and control plants is at least about 10%, preferably at least about 20%, most preferably at least about 30%.
Several parameters can be measured to compare the properties or quality of fibers produced from transgenic plants transformed with MYB nucleic acids and the quality of fibers produced from native plants. These include: 1) fiber length; 2) fiber strength; and 3) fineness of fibers.
A number of methods are known in the art to measure these parameters. See, e.g., U.S. Pat. No. 5,495,070, incorporated herein by reference. For example, instruments such as a fibrograph and HVI (high volume instrumentation) systems can be used to measure the length of fibers. The HVI systems can also be used to measure fiber strength. Fiber strength generally refers to the force required to break a bundle of fibers or a single fiber. In HVI testing, the breaking force is expressed in terms of xe2x80x9cgrams force per tex unit.xe2x80x9d This is the force required to break a bundle of fibers that is one tex unit in size. In addition, fineness of fibers can be measured, e.g., from a porous air flow test. In a porous air flow test, a weighed sample of fibers is compressed to a given volume and controlled air flow is passed through the sample. The resistance to the air flow is read as micronaire units. The micronaire readings reflect a combination of maturity and fineness. Using these and other methods known in the art, one of skill can readily determine the extent of modulation of fiber characteristics or quality in transgenic plants.